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1.
Physiol Rev ; 76(1): 49-67, 1996 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8592732

RESUMO

The cardiac action potential results from the complex, but precisely regulated, movement of ions across the sarcolemmal membrane. Potassium channels represent the most diverse class of ion channels in heart and are the targets of several antiarrhythmic drugs. Potassium currents in the myocardium can be classified into one of two general categories: 1) inward rectifying currents such as IK1, IKACh, and IKATP; and 2) primarily voltage-gated currents such as IKs, IKr, IKp, IKur, and Ito. The inward rectifier currents regulate the resting membrane potential, whereas the voltage-activated currents control action potential duration. The presence of these multiple, often overlapping, outward currents in native cardiac myocytes has complicated the study of individual K+ channels; however, the application of molecular cloning technology to these cardiovascular K+ channels has identified the primary structure of these proteins, and heterologous expression systems have allowed a detailed analysis of the function and pharmacology of a single channel type. This review addresses the progress made toward understanding the complex molecular physiology of K+ channels in mammalian myocardium. An important challenge for the future is to determine the relative contribution of each of these cloned channels to cardiac function.


Assuntos
Miocárdio/metabolismo , Canais de Potássio/genética , Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Arritmias Cardíacas/genética , Clonagem Molecular , Condutividade Elétrica , Coração/fisiologia , Humanos , Dados de Sequência Molecular
2.
Circ Res ; 75(6): 999-1005, 1994 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-7955154

RESUMO

The transient outward current (ITO) is an important repolarizing component of the cardiac action potential. In native cardiac myocytes, ITO is modulated after activation of protein kinase C, although the molecular nature of this effect is not well understood. A channel recently cloned from human ventricular myocardium (Kv1.4, HK1) produces a rapidly inactivating K+ current, which has phenotypic similarities to the 4-aminopyridine-sensitive component of ITO. Therefore, we examined whether this recombinant channel was also modulated by protein kinase C activation by investigating the effects of the diacylglycerol analogue phorbol 12-myristate 13-acetate (PMA) on Kv1.4 K+ current expressed in Xenopus oocytes. At a concentration of 10 nmol/L, PMA caused a biphasic response with an initial increase (14 +/- 4%, mean +/- SEM) in current, which peaked in 14 minutes. This was followed by a significant reduction (40 +/- 11%) in the current within 30 minutes. There was no significant change in cell membrane electrical capacitance with 10 nmol/L PMA (1 +/- 1% decline in 30 minutes), demonstrating that loss of cell membrane surface area did not explain the reduction in K+ current, although cell capacitance did decrease when using a higher concentration of PMA (81 nmol/L). The inactive stereoisomer, 4 alpha-PMA, had no effect on Kv1.4 current, whereas preincubation with the protein kinase inhibitor staurosporine or protein kinase C-selective chelerythrine prevented the effects of PMA. When purified from a stably transfected mammalian cell line by using immunoprecipitation, the channel protein was readily phosphorylated in vitro by purified protein kinase C.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Coração/fisiologia , Miocárdio/metabolismo , Canais de Potássio/fisiologia , Proteína Quinase C/fisiologia , Potenciais de Ação/fisiologia , Alcaloides/farmacologia , Animais , Benzofenantridinas , Membrana Celular/metabolismo , Clonagem Molecular , Ativação Enzimática , Coração/efeitos dos fármacos , Humanos , Miocárdio/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Fenantridinas/farmacologia , Fenótipo , Ésteres de Forbol/farmacologia , Fosforilação , Canais de Potássio/genética , Canais de Potássio/metabolismo , Proteína Quinase C/antagonistas & inibidores , Estaurosporina , Fatores de Tempo , Transfecção , Xenopus
3.
J Neurosci ; 14(3 Pt 2): 1666-76, 1994 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8126562

RESUMO

While combined cloning, mutagenesis, and electrophysiological techniques have provided great insight into K+ channel structure/function relationships, little is known about K+ channel biosynthesis. To examine K+ channel biosynthesis, immune purifications were conducted on Triton X-100 extracts of 35S-met-labeled channels from in vitro translations and transfected mouse L-cells. When Kv1.1 and Kv1.4 were cotranslated in vitro, isoform-specific antisera copurified both proteins even at early time points, suggesting rapid subunit assembly. The non-Shaker Kv2.1 channel did not assemble with Kv1.1 or Kv1.4. Mouse L-cells transfected with Kv1.1 cDNA yielded 1000-4000 functional surface channels, and immune purification from Kv1.1 cells with Kv1.1 antisera produced a 57-59 kDa doublet on SDS-PAGE but not in sham-transfected cells. Immune purification of surface channels isolated both the 57 and 59 kDa proteins, suggesting cell surface channels are represented by two species. Pulse-chase metabolic labeling studies were consistent with a precursor-product relationship with the 57 kDa species giving rise to the 59 kDa protein within several minutes of synthesis. At longer chase times, the 57 kDa species reappeared, indicating both an early precursor and a mature protein ran with identical electrophoretic mobility. Mutation of the extracellular glycosylation site (N207) yielded two proteins at steady state, a 55 kDa core peptide and a 57 kDa species. Lack of glycosylation at N207 had little effect on channel synthesis, turnover, or function. Together these results suggest (1) heteromeric assembly of Shaker-like channels is cotranslational, and (2) N207 glycosylation of Kv1.1 occurs but is not required for subunit assembly, transport, or function.


Assuntos
Encéfalo/metabolismo , Canais de Potássio/metabolismo , Processamento de Proteína Pós-Traducional , Sequência de Aminoácidos , Animais , Glicosilação , Técnicas Imunológicas , Camundongos , Dados de Sequência Molecular , Canais de Potássio/química , Canais de Potássio/genética
4.
Am J Physiol ; 265(5 Pt 1): C1230-8, 1993 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8238476

RESUMO

Voltage-gated K+ channels are involved in regulation of action potential duration and in setting the resting membrane potential in nerve and muscle. To determine the effects of voltage-gated K+ channel expression on processes not associated with electrically excitable cells, we studied cell volume, membrane potential, Na(+)-K(+)-ATPase activity, and alanine transport after the stable expression of the Kv1.4 and Kv1.5 human K+ channels in Ltk- mouse fibroblasts (L-cells). The fast-activating noninactivating Kv1.5 channel, but not the rapidly inactivating Kv1.4 channel, prevented dexamethasone-induced increases in intracellular volume and inhibited Na(+)-K(+)-ATPase activity by 25%, as measured by 86Rb+ uptake. Alanine transport, measured separately by systems A and ASC, was lower in Kv1.5-expressing cells, indicating that the expression of this channel modified the Na(+)-dependent amino acid transport of both systems. Expression of the Kv1.4 channel did not alter alanine transport relative to wild-type or sham-transfected cells. The changes specific to Kv1.5 expression may be related to the resting membrane potential induced by this channel (-30 mV) in contrast to that measured in wild-type sham-transfected, or Kv1.4-transfected cells (-2 to 0 mV). Blocking of the Kv1.5 channel by 60 microM quinidine negated the effects of Kv1.5 expression on intracellular volume, Na(+)-K(+)-ATPase, and Na(+)-dependent alanine transport. These results indicate that delayed rectifier channels such as Kv1.5 can play a key role in the control of cell membrane potential, cell volume, Na(+)-K(+)-ATPase activity, and electrogenic alanine transport across the plasma membrane of electrically unexcitable cells.


Assuntos
Alanina/metabolismo , Potenciais da Membrana/fisiologia , Canais de Potássio/biossíntese , Rubídio/metabolismo , Animais , Transporte Biológico , Clonagem Molecular , DNA Complementar/metabolismo , Fibroblastos/fisiologia , Cinética , Células L , Camundongos , Proteínas Recombinantes/biossíntese , Mapeamento por Restrição , ATPase Trocadora de Sódio-Potássio/metabolismo , Fatores de Tempo , Transfecção , beta-Alanina/análogos & derivados , beta-Alanina/metabolismo
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